Carbapenem-resistant Enterobacteriaceae are an emerging class of multi-drug resistant bacterial pathogens that are either effectively untreatable or only treatable with toxic antimicrobials. Their resistance to carbapenems is especially problematic, as these agents are often the last line of defense against drug- resistant pathogens. Therefore, the CDC now categorizes such carbapenem-resistant Enterobacteriaceae (CRE) in their top antibiotic resistance threat level. New anti-infective strategies are urgently needed. Carbapenemase genes (and resistance to many other antimicrobials) are carried on large, low copy number plasmids. An underlying hypothesis of this proposal is that it should be possible to target these plasmids for eviction, thereby restoring carbapenem susceptibility to these strains. Considered more broadly, this strategy might also be employed to restore susceptibility to many other antimicrobials as well. Therefore, proof of principle is sought for combating carbapenem resistance through plasmid eviction therapy. To accomplish this goal, in the R21 phase, a screening strategy will be developed, validated, and implemented to identify small molecule inhibitors of plasmid maintenance. The screening strategy is based on technology that will allow quantitative assessment of plasmid loss. Specifically, novel transposons will be used to integrate reporter genes into the carbapenemase resistance plasmid and bacterial chromosome of a screening strain, thereby providing an easily measured readout of normalized plasmid number. A mid-size high throughput screen for anti-plasmid agents will then be used to identify potent inhibitors of plasmid maintenance. The inhibitors will be tested for their ability to restore carbapenem susceptibility, i.e., adjunctive antimicrobial activity. In the R33 phase, a larger high throughput screen will be performed to identify additional potent anti-plasmid agents appropriate for further development. Iterative structure-activity relationship studies will then be used to identify analogues with enhanced pharmacological potential. Finally, the activity of select anti-plasmid agents will be characterize in murine infection models to establish in vivo efficacy. Taken together, these experiments should establish the theoretical basis for this adjunctive antimicrobial therapy and its potential use as a new human therapeutic.